Liquid cell and passivated probe for atomic force microscopy and chemical sensing

ABSTRACT

The invention provides a liquid cell for an atomic force microscope. The liquid cell includes a liquid cell housing with an internal cavity to contain a fluid, a plurality of conductive feedthroughs traversing the liquid cell housing between the internal cavity and a dry side of the liquid cell, a cantilevered probe coupled to the liquid cell housing, and a piezoelectric drive element disposed on the cantilevered probe. The cantilevered probe is actuated when a drive voltage is applied to the piezoelectric drive element through at least one of the conductive feedthroughs. A method of imaging an object in a liquid medium and a method of sensing a target species with the liquid cell are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This utility application claims the priority benefit of U.S. ProvisionalApplication No. 60/453,979, filed Mar. 11, 2003.

FIELD OF THE INVENTION

This invention relates generally to atomic force microscopy (AFM) andmethods of imaging thereof. In particular, the invention relates to AFMimaging of samples in liquid, and the detection of specific chemical andbiological species using vibrating cantilevered probes.

BACKGROUND OF THE INVENTION

Atomic force microscopy, also called scanning force microscopy andscanning probe microscopy, has become an important tool for biologicalscience, with significant application to imaging samples such DNA andliving cells in solution. When imaging biological samples, an atomicforce microscope (AFM) is usually operated with a liquid cell, becausethe samples need to remain immersed in order to retain naturalcharacteristics.

Generally atomic force microscopes for biological science employ liquidcell housings that contain a specimen in fluid. A fluid cell for anatomic force microscope is described by Hansma et al. in “Atomic ForceMicroscope with Optional Replaceable Fluid Cell,” U.S. Pat. No.4,935,634 issued Jun. 19, 1990. The optional and replaceableprobe-carrying module includes the provision for forming a fluid cellaround the AFM probe.

An electro-chemical liquid cell for use with an atomic force or scanningtunneling microscope is described by Lindsay et al. in “Scanning ProbeMicroscope for Use in Fluids,” U.S. Pat. No. 5,750,989 issued May 12,1998. A hermetically sealed chamber may be formed around a sample by aseal between the scanner of the microscope and its frame.

Knauss et al. describes a hyperbaric hydrothermal atomic forcemicroscope with a gas pressurized microscope base chamber and a samplecell environment in “Hyperbaric Hydrothermal Atomic Force Microscope,”U.S. Pat. No. 6,437,328 issued Aug. 20, 2002. The AFM images solidsurfaces in liquid or gas that flow within the sample cell at pressuresgreater than normal atmospheric pressure.

Tapping mode AFM has become an important tool, capable ofnanometer-scale resolution on biological samples. The periodic contactwith the sample surface minimizes frictional forces, avoidingsignificant damage to fragile or loosely attached samples. Arepresentative tapping AFM is described by Elings et al. in “TappingAtomic Force Microscope,” U.S. Pat. No. 5,412,980 issued May 9, 1995.

Liquid tapping-mode AFM, also referred to as liquid cyclic-mode AFM, isa scanning probe imaging mode that is suitable for biological imagingand is used frequently to obtain nanometer-scale resolution on fragilespecimens. Liquid tapping-mode AFM helps to minimize friction damagethat is characteristic of contact-mode AFM, to reduce van der Waalsforces, and to eliminate capillary forces between an AFM cantilever tipand a specimen. Tapping mode has been used to image DNA in situ, thefolding and unfolding of individual titin molecules, crystal growth,Langmuir-Blodgett films, polymers, living plant cells, red and whiteblood cells, moving myosin V molecules, and numerous other biologicalsamples.

AFMs in a liquid tapping mode typically employ a cantilever, an externalpiezoelectric oscillator, and an optical displacement-sensing component.An AFM operating in a vibrating, cyclic or tapping mode may use apiezoelectrically actuated microcantilevered probe. Typically, the probeis a micro-electrical-mechanical-system (MEMS) device, micromachinedfrom bulk silicon and silicon-on-insulator (SOI) wafers with apiezoelectric film patterned along a portion of the microcantilever. Atthe free end of the cantilever is a tip with nanometer-scale radius,optimally shaped to probe the sample surface. The microcantilever isdisplaced by voltage applied to the piezoelectric actuator, resulting ina controlled vertical movement of the tip. Control electronics drive themicrocantilever while simultaneously positioning it vertically to trackthe sample topography and follow the surface features. A macro-scaleposition actuator such as a piezotube may be used to null the positionof the cantilever, following the topology of the sample as the probe isscanned over the surface. Smaller AFM cantilevers have been developed,contributing to improvements in the imaging speed of the liquid tappingmode.

Xu et al. describes an AFM with a cantilever tip for probing abiological specimen in “Atomic Force Microscope for BiologicalSpecimens, U.S. Pat. No. 5,874,668 issued Feb. 23, 1999. The cantileveris designed to identify physiologically and pharmacologically importantbiomolecules and their constituent subunits. For example, a cantilevercan be manufactured to be biospecific, allowing the identification ofspecific voltage-sensitive tissues and biomolecules.

A sensor using a cantilever to detect a selected target species isdisclosed by Lee et al. in “Chemical and Biological Sensor Using anUltra-Sensitive Force Transducer,” U.S. Pat. No. 5,807,758 issued Sep.15, 1998. This chemical and biological sensor has a cantilever withattached chemical modifiers capable of undergoing a selective bindinginteraction. A target specimen in contact with the cantilever cangenerate an electric or magnetic field that induces a measurabledeflection. The target molecule may be in liquid phase or in vaporphase.

Another chemical sensor using microcantilevers is described by Thundatin “Microcantilever Detector for Explosives,” U.S. Pat. No. 5,918,263issued Jun. 29, 1999. This apparatus detects explosive vapor phasechemical, employing a cantilever and a heater for increasing the surfacetemperature of the cantilever that causes combustion of the adsorbedexplosive vapor phase chemical. The combustion results in a deflectionand a resonance response of the cantilever.

A magnetically modulated cantilever is described by Han et al. in“Magnetically-Oscillated Probe Microscope for Operation in Liquids,”U.S. Pat. No. 5,753,814 issued May 19, 1998. The invention employs anAC-driven atomic force microscope with a ferrite-core solenoid formodulating the magnetic cantilever. The detection system for themagnetically modulated AC-AFM incorporates AC coupling of the signalfrom the position sensitive detector/beam deflection detector in orderto remove the DC component of the signal. The result is an improveddynamic range over systems using DC coupling.

Attempts have been made to increase the speed of AFM imaging. Anamplitude detection circuit is used to dynamically control thecantilever drive signal in an amplitude domain, as described by Addertonet al. in “Dynamic Activation for an Atomic Force Microscope and Methodof Use Thereof,” U.S. Patent Application 2002/0062684, published May 30,2002.

Lee et al. have used piezoelectric lead-zirconate-titanate (PZT)actuated cantilevers to achieve 1,030 pixels/s and tip speeds of 16μm/s, as disclosed in J. Vac. Sci. & Tech., B 15(4), 1559 (1997).Cantilever probes with a thin integrated film of zinc oxide (ZnO)serving as an actuator have achieved a resonance frequency on the orderof 15 kHz in liquid. The results are faster imaging speeds and improvedtuning capability, as reported by Sulchek et al. in Rev. Sci. Instrum.71(5), 2097 (2000), and by Rogers et al. in Rev. Sci. Instrum. 73(9),3242 (2002). For most biological samples, conventional AFMs can scan atspeeds of a few tens of microns per second, which could require severalminutes to produce a 512×512 pixel image. Thus, the scan rate of an AFMmay be too slow for applications where biological and chemical processesoccur in less than a minute.

Faster measurement times would help shrink the existing separationbetween the time scales of force spectroscopy experiments and the timescales of molecular dynamics calculations. Quicker scan speeds wouldreduce the time spent locating interesting features and would enable thestudy of dynamics occurring in liquid or physiological environments. Animproved method for scanning a specimen that is in a liquid environmentwould scan more quickly and provide better tuning capability thancurrently used AFM cantilever probes. In addition, an improved methodwould provide a real-time imaging tool for studying dynamic phenomena inphysiological conditions.

Therefore, what is needed is a structure and a method for quickerimaging times for contact and tapping mode atomic force microscopy inliquid, and for sensing target chemical and biological species inliquid, overcoming the deficiencies and obstacles described above.

SUMMARY OF THE INVENTION

One aspect of the invention provides a liquid cell for an atomic forcemicroscope. The liquid cell includes a liquid cell housing with aninternal cavity to contain a fluid, a plurality of conductivefeedthroughs traversing the liquid cell housing between the internalcavity and a dry side of the liquid cell, and a cantilevered probe,which is coupled to the liquid cell housing with at least a portion ofthe cantilevered probe located inside the internal cavity. The liquidcell includes a piezoelectric drive element disposed on the cantileveredprobe. The cantilevered probe is actuated when a drive voltage isapplied to the piezoelectric drive element through at least one of theconductive feedthroughs.

The liquid cell may include an optical window in the liquid cellhousing, so that a beam of light may traverse the optical window tooptically monitor the cantilevered probe. The liquid cell may include aninlet port connected between the internal cavity and the dry side of theliquid cell, so that fluid may be injected into the internal cavity ofthe liquid cell housing. A passivation layer may be disposed onto thepiezoelectric drive element to electrically isolate the piezoelectricdrive element when a conductive fluid is placed in the internal cavityof the liquid cell housing. An atomic probe tip may be coupled to a freeend of the cantilevered probe. A treated section may be coupled to thecantilevered probe. The treated section causes a deflection of thecantilevered probe or a shift in a resonant frequency of thecantilevered probe when exposed to a target species. A piezoresistivesense element may be coupled to the cantilevered probe to sense bendingof the cantilevered probe. The liquid cell may include a probe heatercoupled to the cantilevered probe that is heated when a heater voltageis applied to at least one conductive feedthrough that is electricallyconnected to the probe heater.

Another aspect of the invention provides a method of imaging an objectin a liquid medium. A liquid cell is positioned adjacent to the object,the liquid cell including a liquid cell housing with an internal cavityand a plurality of conductive feedthroughs connected between theinternal cavity and a dry side of the liquid cell. The position of anatomic probe tip, which is coupled to a free end of a cantileveredprobe, is measured when the atomic probe tip contacts the object, thecantilevered probe being coupled to the liquid cell housing and aportion of the cantilevered probe being located inside the internalcavity. An image of the object is generated based on the measuredposition of the atomic probe tip when the atomic probe tip is scannedacross the object.

An excitation voltage may be applied through at least one conductivefeedthrough to a piezoelectric drive element disposed on thecantilevered probe, the excitation voltage being applied at a frequencynear a resonant frequency of the cantilevered probe to tap the atomicprobe tip against the object. A fluid may be injected onto the objectthrough an inlet port, which is connected between the internal cavityand the dry side of the liquid cell.

Another aspect of the invention provides a method of sensing a targetspecies with a liquid cell. A cantilevered probe is driven with anexcitation voltage. A first deflection amplitude of the cantileveredprobe is measured with a sense element coupled to the cantileveredprobe. A treated section of the cantilevered probe is exposed to atarget species. A second deflection amplitude of the cantilevered probeis measured, and the target species is determined based on the firstdeflection amplitude measurement and the second deflection amplitudemeasurement. A first frequency of the cantilevered probe may be measuredwith the sense element coupled to the cantilevered probe. The treatedsection of the cantilevered probe is exposed to the target species. Asecond frequency of the cantilevered probe is measured, and the targetspecies is determined based on the first frequency measurement and thesecond frequency measurement.

A fluid including the target species may be injected into the interiorcavity of the liquid cell through an inlet port, which is connectedbetween the internal cavity and the dry side of the liquid cell. Anatomic probe tip coupled to a free end of the cantilevered probe may betapped against a mechanical stop, which may be coupled to a base end ofthe cantilevered probe.

The current invention is illustrated by the accompanying drawings ofvarious embodiments and the detailed description given below. Thedrawings should not be taken to limit the invention to the specificembodiments, but are for explanation and understanding. The detaileddescription and drawings are merely illustrative of the invention ratherthan limiting, the scope of the invention being defined by the appendedclaims and equivalents thereof. The forgoing aspects and other attendantadvantages of the present invention will become more readily appreciatedby the detailed description taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention are illustrated by theaccompanying figures, wherein:

FIG. 1 illustrates a liquid cell for an atomic force microscope, inaccordance with one embodiment of the current invention;

FIG. 2 illustrates a top view of a cantilevered probe, in accordancewith one embodiment of the current invention;

FIG. 3 illustrates a perspective view of a cantilevered probe, inaccordance with another embodiment of the current invention;

FIG. 4 is a flow diagram of a method for imaging an object in a liquidmedium, in accordance with one embodiment of the current invention; and

FIG. 5 is a flow diagram of a method for sensing a target species with aliquid cell, in accordance with one embodiment of the current invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a liquid cell for an atomic force microscope, inaccordance with one embodiment of the current invention at 100. Liquidcell 100 includes a liquid cell housing 110, one or more conductivefeedthroughs 120, a cantilevered probe 130 coupled to liquid cellhousing 110, and a drive element 140 disposed on cantilevered probe 130.At least a portion of cantilevered probe 130 is located inside aninternal cavity 112. Liquid cell 100 may be used to image an object or asurface, or to sense a target chemical or biological species. Liquidcell 100 may be used, for example, in a handheld system for sensing atarget chemical or biological species, or in an atomic force microscope(AFM) for imaging objects and surfaces. Liquid cell 100 may hold one ormore cantilevered probes 130, for example, arranged in an array for highthroughput imaging, or in an array with individually treated probes fordetecting one or more target species. Multiple cantilevered probes 130may be configured to provide additional sensitivity or selectivity tomultiple chemical species within the same liquid cell 100, or to cancelout common-mode effects.

Drive element 140 on cantilevered probe 130 may be operated at a voltagebelow the electrolysis limit of a fluid encompassing drive element 140to allow operation in the fluid without a protective passivation layer.Alternatively, cantilevered probe 130 may be operated at a highervoltage with a protective coating covering at least drive element 140.

Cantilevered probe 130 may be driven into oscillation with a periodicvoltage, may be deflected with a constant or non-periodic voltage, ormay be driven into oscillation with a period voltage while beingdeflected with a constant or non-periodic voltage by drive element 140.Forced oscillations of cantilevered probe 130 are useful for tappingmode atomic force microscope imaging and frequency-shift chemicalsensing. Deflections of cantilevered probe 130 with application ofconstant or non-periodic voltage are useful for maintaining a constantforce at high speeds in various modes of atomic force microscope imagingand for taking force-distance measurements.

Liquid cell housing 110 may be formed from plastic, metal, or anysuitable material for housing cantilevered probe 130 and conductivefeedthroughs 120. For example, the liquid cell housing may comprise amaterial such as an acrylic, polymethyl methacrylate (PMMA), glass,quartz, a thermoplastic polymer, a thermoset polymer, an insulatingmaterial, or a transparent material. Liquid cell housing 110 includesinternal cavity 112 that may contain, for example, a non-conductivefluid such as a isopropyl alcohol or a conductive fluid such as salinesolution. Other fluids that may be contained in internal cavity 112include gases such as air. Internal cavity 112 is generally opened onthe bottom to allow a sample to be measured while it is in a fluid, thefluid being partially trapped inside internal cavity 112 by meniscusforces, or the fluid being injected into internal cavity 112 through aninlet port 180. Inlet port 180 is connected between internal cavity 112and dry side 114 of liquid cell 100. Internal cavity 112 may be closedon the bottom to entrap a sample gas or liquid within internal cavity112 during chemical detection and analysis.

The gas or liquid may be injected from a fluid source 182. The effluentmay be exhausted through an outlet port 184 to a fluid receiver 186,vented to the atmosphere, or otherwise contained, discarded orrecirculated. A target chemical or biological species, typically carriedin the air, in a liquid, or in a controlled gas environment, entersinterior cavity 112 of liquid cell 100 through inlet port 180 and mayexit through outlet port 184. Pumps, valves, or other fluidic controldevices may be included to aid in the transport of chemical orbiological fluids to and from interior cavity 112 of liquid cell 100,and seals around inlet port 180 and outlet port 184 may be used tominimize leakage.

Liquid cell 100 may also contain suitable connectors, photodetectors andother elements for controlling and monitoring cantilevered probe 130.Liquid cell 100 may include filters, scrubbers, and other mediatreatment elements to aid in the imaging of objects and surfaces, and inthe detection of target chemical and biological species.

Conductive feedthroughs 120 traverse liquid cell housing 110 betweeninternal cavity 112 and a dry side 114 of liquid cell 100, and provideelectrical connectivity between outside connections to printed circuitboards, sockets, AFM heads, and other electronic circuitry and internalconnections to cantilevered probe 130 and other electronic devicescontained in liquid cell 100. Conductive feedthroughs 120 may comprise,for example, pins, posts, wires, connectors, plated feedthroughs,insulated metal pins, or any suitable feedthrough mechanism available inthe art, and may be arranged to readily accommodate the externalconnections. In addition to electrical connectivity, conductivefeedthroughs 120 may provide mechanical connectivity between liquid cell100 and an externally coupled device. Conductive feedthroughs 120 andconnecting wires may be passivated on the liquid side of liquid cell100, and electrical signals may run through liquid cell housing 110 todry side 114 and control circuitry 150. Conductive feedthroughs 120 andconnecting wires or conductors may be passivated, for example, with asilicone rubber, a silicone gel, an epoxy, or a suitable insulatingcoating.

Cantilevered probe 130 is typically a relatively thin beam clipped,screwed, or otherwise mounted securely to liquid cell housing 110 at abase end 132 of cantilevered probe 130. A free end 134 of cantileveredprobe 130 is generally free to vibrate and bend when driven, forexample, by drive element 140, by continuous or intermittent contactwith a surface or object to be imaged, or when exposed to a targetchemical or biological species. An atomic probe tip 136 may be coupledto free end 134 of cantilevered probe 130.

Cantilevered probe 130 may comprise a layer of silicon, polysilicon,silicon nitride, a metal film, a metal sheet, a zinc oxide (ZnO) film, alead-zirconate-titanate (PZT) film, silicon dioxide, a polymeric layer,or combinations thereof. Cantilevered probe 130 may comprise apiezoelectric drive element 140, which may be actuated when a drivevoltage is applied to piezoelectric drive element 140 through at leastone of the conductive feedthroughs 120. For example, cantilevered probe130 may comprise a layer of silicon with a thin layer of zinc oxide orPZT disposed on one side of the silicon layer. Zinc oxide may bedeposited on cantilevered probe 130 using, for example, a sputteringprocess. PZT may be deposited on cantilevered probe 130 using, forexample, a sol-gel process. In another example, cantilevered probe 130comprises a layer of silicon nitride with a patterned piezoelectric filmon one side of the silicon nitride layer. Two thin layers of a metalsuch as gold or platinum are positioned on each side of the patternedpiezoelectric film, providing electrical contact to the piezoelectricfilm. In another example, cantilevered probe 130 comprises a layer ofsingle-crystal silicon, a piezoresistive layer such as depositedpolycrystalline silicon, and a dielectric layer such as silicon dioxidepositioned between the single-crystal silicon layer and thepiezoresistive layer. In another example, a piezoresistor is formed inor near a surface of a single-crystal silicon cantilever. In anotherexample, cantilevered probe 130 comprises a thin piece of metal such assteel upon which a piezoelectric film is deposited. In another example,cantilevered probe 130 comprises a polymeric layer such aspolymethylmethacrylate (PMMA), polyimide, or a plastic.

Atomic probe tip 136 may be coupled to free end 134 of cantileveredprobe 130 to scan a sample surface or object being imaged, or to tapagainst the sample surface or object when cantilevered probe 130 isoscillated. Atomic probe tip 136 may be integrally formed withcantilever probe 130. For example, atomic probe tip 136 and cantileveredprobe 130 can be formed from thin films such as a silicon nitride filmthat are deposited conformally onto a silicon wafer surface and into anetch pit formed in the silicon wafer surface, such that the siliconnitride film is subsequently patterned and etched to form cantileveredprobe 130 with atomic probe tip 136. In another example, atomic probetip 136 may be formed from silicon or silicon dioxide by selectivepatterning and etch back steps, as is known in the art. In anotherexample, a monolithic atomic probe tip 136 is attached to cantileveredprobe 130 using micromanipulators and standard adhesives. Atomic probetip 136 may be formed from tungsten, carbon nanotubes, diamond, or anyrelatively hard material that can be formed into a tip or asmall-diameter cylinder, and formed on or attached to cantilevered probe130. Atomic probe tip 136 provides a small, pointed, contact surface fortapping against an object, surface, or a mechanical stop 350 (FIG. 3)when cantilevered probe 130 is oscillated. A small contact surface isgenerally desired for higher spatial resolution and for minimizingstiction or other forces that may cause atomic probe tip 136 toinadvertently stick when oscillated.

Cantilevered probe 130 may be driven into oscillation with drive element140 so that oscillating cantilevered probe 130 is tapped against anobject, surface, or mechanical stop. Amplitudes of oscillation andchanges in the oscillation amplitude are measured while an object orsurface is scanned, or after cantilevered probe 130 is exposed to achemical or biological species. A target chemical or biological speciesmay be determined based on oscillation amplitudes of cantilevered probe130 that are measured by sense element 142 when a treated section 144coupled to cantilevered probe 130 is exposed to a target chemical orbiological species. Treated section 144 may cause a deflection ofcantilevered probe 130 or a shift in a resonant frequency ofcantilevered probe 130 when exposed to a target species.

Cantilevered probe 130 includes a treated section 144. Treated section144 may comprise, for example, a selective coating or a patterned filmon cantilevered probe 130. Cantilevered probe 130 may comprise, forexample, a bimorph cantilevered probe where one layer of the bimorphcomprises a silicon layer and a second layer comprises a chemicallysensitive material.

Treated section 144 responds when exposed to a target chemical orbiological species. Treated section 144 may respond by absorbing,adsorbing, or otherwise reacting to a target chemical or biologicalspecies. When exposed to a target chemical or biological species,treated section 144 may increase or decrease in mass, or become morerigid or less rigid. In one example, treated section 144 comprises apatterned layer of gold. When exposed to mercury, the two elements reactto form an amalgam. The gold-mercury amalgam adds mass to cantileveredprobe 130 and therefore tends to decrease the resonant frequency ofcantilevered probe 130. Amalgam formation, however, increases themechanical stiffness of cantilevered probe 130, thereby increasing itsnatural resonant frequency. The two effects tend to cancel each other,though one effect can be made dominant by careful selection andplacement of treated section 144 on cantilevered probe 130. Since thelayers comprising cantilevered probe 130 are typically thin, anyresponse by treated section 144 to a target chemical or biologicalspecies will tend to bend and curl cantilevered probe 130 either up ordown. Minimal displacement of free end 134 of cantilevered probe 130 dueto bending occurs when a small section close to free end 134 is treated.Increased displacement of free end 134 of cantilevered probe 130 occurswhen treated section 144 is located near base end 132. Maximumdisplacement of free end 134 of cantilevered probe 130 occurs whentreated section 144 comprises an entire side of cantilevered probe 130,such as when one side of cantilevered probe 130 is coated with a thinfilm of gold. Because treating the cantilevered probe symmetrically onboth sides tends to negate the bending effects, a treatment ofcantilevered probe 130 is generally applied to one side or the other.

Vibrations or oscillations of cantilevered probe 130 generally occursymmetrically above and below the static displacement profile or neutralposition of cantilevered probe 130, whether cantilevered probe 130 isbent upwards, downwards, or is straight. Bending effects with exposureto a target chemical or biological species are likely to occur much moreslowly than individual oscillations of cantilevered probe 130, and arereferred to as quasi-static bending.

Treated section 144 of cantilevered probe 130 may comprise a coatingsuch as, for example, a gold layer, a palladium layer, analcohol-absorbent polymer, a water-absorbent material, achemical-sensitive layer, a biosensitive material, or a thiol. Treatedsection 144 of cantilevered probe 130 may be selected such that theoscillating cantilevered probe can detect and is sensitive to mercury,hydrogen, an alcohol, water vapor, a chemical element, a chemicalcompound, an organic material, an inorganic material, a biologicalmaterial, a DNA strand, a bioactive agent, a toxin, or any such chemicalor biological material that can be detected with a treated cantileveredprobe.

Treated section 144 of cantilevered probe 130 may comprise, for example,a thin film covering one side of cantilevered probe 130, such as the topor bottom of cantilevered probe 130, or a section of either side. In oneembodiment, treated section 144 of cantilevered probe 130 comprises apatterned thin film located near base end 132 of cantilevered probe 130.Treated section 144 of cantilevered probe 130 may be selectivelypatterned and etched to achieve large displacements with chemical orbiological exposure, yet still may allow room for other probe elementssuch as drive element 140 and sense element 142 to be placed oncantilevered probe 130.

Drive element 140 may comprise any suitable actuation mechanism such as,for example, a piezoelectric drive element, an electrostatic driveelement, a thermal drive element, a magnetic drive element, or othersuitable drive mechanism as is known in the art. Drive element 140coupled to cantilevered probe 130 may comprise, for example, apiezoelectric film disposed near base end 132 of cantilevered probe 130.When drive voltages are applied to the piezoelectric film, cantileveredprobe 130 bends according to the level of the voltage, and the forcesand moments generated by the piezoelectric film. When an oscillatingvoltage is applied to the piezoelectric film, cantilevered probe 130 maybe driven at or near a natural resonant frequency and may achieve a muchlarger amplitude than a statically driven probe. The achieved deflectionamplitude may depend, for example, on the frequency and mode ofoscillation, the internal damping of the probe and applied films, andviscous damping due to gas or liquids surrounding the probe. Driveelement 140 and sense element 142 may comprise, for example, a unitarypiezoelectric element coupled to the cantilevered probe.

Sense element 142 generates a signal based on the static and dynamicdeflections of cantilevered probe 130. The generated signals may be usedto determine static deflections, quasi-static deflections, oscillationamplitudes and frequencies of cantilevered probe 130. A piezoelectricfilm such as a zinc oxide film or a PZT layer may be used to generate asignal when cantilevered probe 130 is displaced or vibrated. The samepiezoelectric film may be used to drive cantilevered probe 130 intooscillation, as well as to sense the displacements, sometimes referredto as self-sensing.

Sense element 142 may comprise, for example, a piezoelectric senseelement, an optical sense element, a piezoresistive sense element, anelectrostatic or capacitive sense element, a magnetic sense element, orany suitable sense element as is known in the art. Sense element 142 maycomprise, for example, a light source 160 for directing an incident beamof light 164 through an optical window 170 in liquid cell housing 110onto cantilevered probe 130, and a photodetector 162 for detecting areflected beam of light 166 reflected from cantilevered probe 130 andtraversing optical window 170, whereby frequencies and amplitudes ofoscillations can be measured with photodetector 162 and cantileveredprobe 130 can be optically monitored. Optical window 170 may also beused to view an object or surface when imaging. The angle of opticalwindow 170 may be selected to compensate for the refraction of beam oflight 164 from a laser as beam of light 164 enters and leaves a liquidmedium within liquid cell 100 so that alignment with photodetector 162is maintained.

Light source 160 and associated optics such as mirrors and lenses directincident beam of light 164 onto a surface of cantilevered probe 130.Light source 160, for example, may comprise a laser or a laser diode andcollimating lenses for generating a well-defined light beam. Incidentbeam of light 164, for example, may comprise coherent laser light at apredefined wavelength that is focused and positioned near the free endof cantilevered probe 130. Photodetector 162 may comprise, for example,a position-sensitive detector (PSD), a photodiode, a photodiode array,or a photodetector array. Photodetector 162 may include a filter, forexample, that filters out stray light and transmits light from reflectedbeam of light 166. Photodetector 162 detects reflected beam of light 166and provides a measure of the oscillation amplitude of cantileveredprobe 130 by detecting changes in position and in light intensity of thereflected light. Other optical sense elements may be employed, such asdiffraction gratings attached to free end 134, or interferometrictechniques between a surface of cantilevered probe 130 and a referenceoptical surface.

Cantilevered probe control circuitry 150 comprises circuits andelectronic devices to drive cantilevered probe 130 into oscillation andto measure deflection amplitudes of the oscillating cantilevered probe130. Control and monitoring of the cantilevered probe and positioningelements may be done with control circuitry 150. Control circuitry 150may include, for example, drive circuitry 152 and sense circuitry 154.Drive circuitry 152 and sense circuitry 154 cooperate to drivecantilevered probe 130 into oscillation using any suitable drive element140 such as a piezoelectric drive element, an electrostatic driveelement, a thermal drive element, or a magnetic drive element. Drivecircuitry 152 and sense circuitry 154 cooperate to measure deflectionamplitudes of oscillating cantilevered probe 130 with any suitable senseelement 142 such as an optical sense element, a piezoelectric senseelement, a piezoresistive sense element, a capacitive sense element, ora magnetic sense element.

Control circuitry 150 may include, for example, drive circuitry 152 andsense circuitry 154 that contain suitable electronic circuits to drivecantilevered probe 130 into oscillation with a drive element 140 mountedon cantilevered probe 130, and to measure a signal from a separatepiezoelectric sense element 142 when cantilevered probe 130 isoscillating.

In another example, control circuitry 150 includes drive circuitry 152and sense circuitry 154 that drive cantilevered probe 130 intooscillation with a piezoelectric drive element 140 mounted oncantilevered probe 130, along with electronic circuits for directingincident beam of light 164 onto a surface of oscillating cantileveredprobe 130 and to detect reflected beam of light 166 when the beam oflight is reflected from the surface of the oscillating cantileveredprobe 130.

In another example, control circuitry 150 includes drive circuitry 152and sense circuitry 154 that drive cantilevered probe 130 intooscillation with a unitary piezoelectric element mounted on cantileveredprobe 130, and that sense oscillation amplitudes of cantilevered probe130 with the same unitary piezoelectric element.

In another example, control circuitry 150 includes drive circuitry 152and sense circuitry 154 that drive cantilevered probe 130 intooscillation with thermal drive element coupled to cantilevered probe 130and that sense oscillation amplitudes of cantilevered probe 130 with apiezoresistor formed in cantilevered probe 130.

In another example, control circuitry 150 includes heater circuitry 156that controls power to a probe heater coupled to cantilevered probe 130.A heater voltage may be applied to the probe heater through one or moreconductive feedthroughs 120.

Control circuitry 150 may also include positioning circuitry to adjustthe position of a reference surface, also referred to as a mechanicalstop, when engaging the cantilevered probe and when operatingoscillating cantilevered probe 130 in an open-loop or closed-loop mode.Control circuitry 150 may have an interface to a control computer.

A control computer may be interfaced with control circuitry 150 tocontrol the operations and functions of liquid cell 100. The controlcomputer may include or have access to one or more databases forcollecting, analyzing, and storing data from liquid cell 100. Thecontrol computer may contain suitable hardware and software for imagingan object or a sample surface, or for determining a target chemical orbiological species based on oscillation amplitudes or measuredfrequencies of cantilevered probe 130. The control computer may benetworked.

Although liquid cell 100 is depicted as an imaging head for an AFM, thepresent invention does not require the AFM to drive and sensecantilevered probe 130, particularly when used for chemical andbiological sensing applications. With portable devices, the scanning andsample placement systems can be omitted. In one embodiment, liquid cell100 may be used in a dual mode as part of an AFM and as a chemical orbiological sensor.

FIG. 2 illustrates a top view of a cantilevered probe, in accordancewith one embodiment of the present invention at 200. Cantilevered probe230 includes a drive element 240 located near a base end 232 ofcantilevered probe 230. Cantilevered probe 230 is generally attached toa liquid cell housing near base end 232 of cantilevered probe 230. Afree end 234 of cantilevered probe 230 may vibrate and move freely inresponse, for example, to excitation by drive element 240, when scannedacross an object or a surface, or when exposed to a target species.Drive element 240 may comprise, for example, a piezoelectric driveelement, an electrostatic drive element, a thermal drive element, or amagnetic drive element.

Cantilevered probe 230 may include an atomic probe tip 236 coupled tofree end 234 of cantilevered probe 230. A treated section 244 may becoupled to cantilevered probe 230 to cause, for example, a deflection ofcantilevered probe 230 or a shift in a resonant frequency ofcantilevered probe 230 when treated section 244 is exposed to a targetspecies. A piezoresistive sense element 256 or other suitable senseelement such as a piezoelectric sense element or a magnetic senseelement may be coupled to cantilevered probe 230 to sense bending ofcantilevered probe 230. A magnetic sense element, such as one or moreloops of wire, may be electrically connected to external sense circuitrythrough one or more conductive feedthroughs in the liquid cell. A probeheater 258 may be coupled to cantilevered probe 230 to heat cantileveredprobe 230 when a heater voltage is applied to probe heater 258. Probeheater 258 may comprise, for example, a resistive heater formed in or oncantilevered probe 230. Probe heater 258 may be used to heat thecantilevered probe to an elevated temperature for initializing orre-initializing treated section 244. Probe heater 258 may be used, forexample, to initiate or cause a chemical reaction on cantilevered probe230.

Cantilevered probe 230 may have a rectangular shape, though other shapesmay be suitably used such as a pointed cantilever, a V-shapedcantilever, a triangular-shaped cantilever, or a dual-arm cantilever.Cantilevered probe 230 may be arranged in an array of cantileveredprobes, the cantilevers being all identical, all different, or somecombination thereof. An array of cantilevered probes 230 may be attachedto a common base. The array of cantilevered probes 230 may be driven andsensed, for example, with a unitary piezoelectric element coupled toeach cantilevered probe. In one embodiment, the unitary piezoelectricelements in the array may be connected in series. The series-connectedpiezoelectric elements in the array may be driven with as few as twoelectrical connections to the piezoelectric element array. In this case,scanning the drive voltage through a range of frequency can excite andsense one probe at a time, allowing interrogation of any probe in thearray while minimizing the number of electrical connections required. Inanother configuration, the piezoelectric elements in the array areconnected in parallel, such that as few as two electrical connectionsmay be used to drive and sense the probes and that failure of one probedoes not prevent others from operating. In another configuration, thearray of piezoelectric elements is connected in a series-parallelarrangement.

In an alternative embodiment, cantilevered probe 230 is attached at eachend, with the center of cantilevered probe 230 free to vibrate. Atomicprobe tip 236 may be attached at or near the center of the probe. Inanother embodiment, cantilevered probe 230 is attached on all sides in adiaphragm or membrane configuration, with atomic probe tip 236 locatedat or near the center of the diaphragm. In another embodiment,cantilevered probe 230 in a cantilevered, doubly supported or diaphragmconfiguration has two or more atomic probe tips 236. Multiple probe tipsmay be used, for example, to preferentially excite specific resonantmodes or to provide additional information for detection of chemical orbiological materials.

Electrical connections to drive element 240, piezoresistive senseelement 256, probe heater 258, and other drive or sense elementsincluded on cantilevered probe 230 may be connected to conductivefeedthroughs in the liquid cell through wire bonds or other wiringconnected between the conductive feedthroughs and bond pads 224 oncantilevered probe 230. A passivation layer disposed on drive element240 may have openings 226 to allow electrical connections to bond pads224. Openings 226 may have been formed, for example, by patterning andwet or dry etching during fabrication of cantilevered probe 230.

FIG. 3 illustrates a perspective view of a cantilevered probe, inaccordance with another embodiment of the present invention at 300. Acantilevered probe 330 contains a piezoelectric drive element 340.Piezoelectric drive element 340 is typically located on a surface near abase end 332 of cantilevered probe 330. Piezoelectric drive element 340may comprise a piezoelectric material such as zinc oxide, lead zirconatetitanate, polyvinylidene fluoride, or a piezoelectric film. An upperelectrode 342 and a lower electrode 344 generally sandwich thepiezoelectric material, such that excitation voltages applied to theelectrodes generate an expansion or contraction of cantilevered probe330 to induce bending. Alternately, the piezoelectric material may beused as a sense element, detecting bending, vibrations and displacementsof a free end 334 of cantilevered probe 330. An atomic probe tip 336 maybe coupled near free end 334 of cantilevered probe 330 to aid in theimaging process.

A passivation layer 346 may be disposed onto piezoelectric drive element340 to electrically isolate piezoelectric drive element 340 when, forexample, a conductive fluid is placed in the internal cavity of a liquidcell housing including cantilevered probe 330. Passivation layer 346covers upper electrode 342 and any exposed surfaces of lower electrode344 such as the sidewalls. Passivation layer 346 prevents shorting,arcing, or any parasitic leakage paths between upper electrode 342 andlower electrode 344 that would degrade the performance of piezoelectricdrive element 340. To minimize or reduce shorting or arcing betweenelectrodes 342 and 344, either upper electrode 342, lower electrode 344or both may be smaller than piezoelectric drive element 340 to reducethe possibility that a portion of either electrode 342 and 344 couldtouch the other. Lower electrode 344 may be smaller than piezoelectricdrive element 340 to prevent direct exposure to a conducting liquid inthe fluid cell.

Passivation layer 346 may serve to coat and protect piezoelectric driveelement 340 from conductive liquids and permeation of the piezoelectricmaterial, and to eliminate parallel conducting paths between theelectrodes. Passivation layer 346 may comprise, for example, siliconnitride, silicon dioxide, parylene, Teflon, an insulating polymer, or aninsulating material. Upper electrode 342 and lower electrode 344 aretypically routed via metal traces to bond pads 324 on cantilevered probe330. Bond pad openings 326 may be formed in passivation layer 346 toexpose bond pads 324 for external electrical connection, for example,with wire bonds or other wiring technique. Passivation layer 346 may bedeposited, for example, simultaneously on multitude cantilevered probesat the wafer level, at the die level, on individual cantilevered probes,or after mounting cantilevered probe 330 into the liquid cell.Passivation coatings using conformal coatings such as parylene C may beapplied. Plasma cleaning and other cleaning techniques may be used toimprove adhesion and reduce pinholes and other defects. Masking andetching steps for piezoelectric drive element 340 may be utilized tominimize or eliminate overhanging metal layers that are difficult topassivate.

The wiring between cantilevered probe 330 and conductive feedthroughs inthe liquid cell are generally passivated, using commercially availablecoatings and coating techniques such as RTV, silicone rubber, siliconegel, an epoxy, an insulating polymer or an insulating material.

FIG. 4 shows a flow diagram of a method for imaging an object in aliquid medium, in accordance with one embodiment of the presentinvention at 400. Method of imaging 400 includes various steps to imagean object in a liquid medium.

An object to be imaged is positioned adjacent to a liquid cell, as seenat block 410. The liquid cell includes a liquid cell housing with aninternal cavity and a plurality of conductive feedthroughs connectedbetween the internal cavity and a dry side of the liquid cell. Theobject may be, for example, a substrate or a surface to be imaged, anobject located on a substrate or a surface, or any object that can bepositioned adjacent the liquid cell and imaged. A substrate such as aglass slide or a semiconductor wafer may be placed, for example, underthe liquid cell with a small gap between the bottom of the liquid celland the substrate. The substrate may be positioned, for example, with astage coupled to an atomic force microscope (AFM). The stage may beused, for example, to position the object to be imaged close to or incontact with an atomic probe tip coupled to the cantilevered probe.

Fluid may be injected onto the object, as seen at block 420. The fluidmay be injected onto the object through an inlet port connected betweenthe internal cavity and the dry side of the liquid cell. The fluid forimaging a biological sample may be, for example, a saline solution, asolvent, water, a conductive fluid, a non-conductive fluid, water, or agas. The sample may be already immersed in a fluid when the object isplaced adjacent to the liquid cell, and the fluid may be replenished asneeded through the inlet port. Excess fluid can be removed from theinterior cavity of the liquid cell from under the liquid cell or throughan outlet port, though generally the fluid is adequately contained withsurface tension and meniscus forces in the vicinity of the object beingimaged. In some cases, only a few drops of liquid are needed to coverthe object while being imaged.

An excitation voltage may be applied to a drive element disposed on thecantilevered probe, as seen at block 430. The drive element maycomprise, for example, a piezoelectric drive element, a thermal driveelement, or a magnetic drive element. The excitation voltage may beapplied through one or more conductive feedthroughs to the driveelement.

The excitation voltage may include a topographical component. Thetopographical component may be adjusted to maintain the cantileveredprobe in a constant force mode or a constant amplitude mode when imagingin the contact mode. Feedback from a sense element on the probe or anoptical sense mechanism using a beam of light may be applied to thedrive element on the cantilevered probe to maintain constant force onthe atomic probe tip during scanning. In the constant amplitude mode,feedback may be applied to the drive element to maintain thecantilevered probe at constant amplitude as the object or surface isbeing scanned. Alternatively, feedback may be applied to a piezotubecoupled between the base of the cantilevered probe and the object orsurface to maintain the cantilevered probe in a constant amplitude modeor a constant force mode while scanning. The topological component maybe used to deflect the cantilevered probe when initially contacting theobject or surface. The feedback may be applied to the piezotube inconjunction with the drive element.

The excitation voltage may be applied at a frequency near a resonantfrequency of the cantilevered probe tip to tap the cantilevered probeagainst the object when operating in a tapping mode. A topographicalcomponent may be combined with the oscillating voltage and applied tothe drive element.

The object to be imaged is scanned, as seen at block 440. The object maybe scanned using, for example, a piezotube coupled to the cantileveredprobe and to the liquid cell. In another example, a stage that holds thesample being imaged may be moved. Scanning may occur, for example, withrepeated traces across the object with a small lateral translation ateach consecutive trace.

The position of the atomic probe tip may be measured, as seen at block450. The position of the atomic probe tip coupled to the free end of thecantilevered probe is measured when the atomic probe tip contacts theobject. The cantilevered probe is coupled to the liquid cell housing anda portion of the cantilevered probe is located inside the internalcavity of the liquid cell.

In one example, the position of the atomic probe tip is measured with apiezoelectric sense element disposed on the cantilevered probe. Thepiezoelectric sense element may be electrically connected to externalsense circuitry through one or more conductive feedthroughs in theliquid cell. The piezoelectric sense element may include a material suchas zinc oxide, lead zirconate titanate, polyvinylidene fluoride, or apiezoelectric film. The piezoelectric sense element may be passivated toelectrically isolate the piezoelectric sense element when, for example,a conductive fluid is placed in the internal cavity of the liquid cell.The piezoelectric sense element may be passivated with a material suchas silicon nitride, silicon dioxide, parylene, Teflon, an insulatingpolymer, or a suitable insulating material.

In another example, the position of the atomic probe tip is measuredwith a piezoresistive sense element coupled to the cantilevered probe.The piezoresistive sense element may be electrically connected toexternal sense circuitry through one or more conductive feedthroughs inthe liquid cell. The piezoresistive sense element may be passivated.

In another example, the position of the atomic probe tip is measuredwith a magnetic sense element coupled to the cantilevered probe. Themagnetic sense element, such as one or more loops of wire, may beelectrically connected to external sense circuitry through one or moreconductive feedthroughs in the liquid cell. The magnetic sense elementmay be passivated.

In the tapping mode, an excitation voltage generally at or near aresonant frequency of the cantilevered probe is applied to the driveelement to tap the atomic probe tip against the object. The position ofthe atomic probe tip is measured to determine the local height of theobject and the x-y coordinate at the point of measurement. The x-ycoordinates may be determined, for example, from the stage position orextracted from scanning voltages applied to the piezotube. The positionof the atomic probe tip may be measured, for example, with apiezoelectric sense element disposed on the cantilevered probe. Inanother example, a piezoelectric sense element and a piezoelectric driveelement comprise the same piezoelectric element on the cantileveredprobe. In another example, the position of the atomic probe tip ismeasured with a piezoresistive sense element coupled to the cantileveredprobe. The piezoresistive sense element provides a change in resistancerelated to the bending of the cantilevered probe. In another example,the position of the atomic probe tip is measured with a beam of lightstriking a photodetector after being reflected from at least a portionof the cantilevered probe. Measurements of the atomic probe tip positionmay be combined with the position of the piezotube to generate an image.

An image of the object is generated, as seen at block 460. The image isgenerated from the measured positions of the atomic probe tip when theatomic probe tip is scanned across the object. The images may be storedelectronically, and may be scaled, enhanced, colored, cropped, stitchedor otherwise modified as desired.

FIG. 5 shows a flow diagram of a method for sensing a target specieswith a liquid cell, in accordance with one embodiment of the presentinvention at 500. Target species sensing method 500 includes varioussteps to detect and determine a target biological or chemical species byusing a cantilevered probe.

The cantilevered probe is driven, as seen at block 510. The cantileveredprobe is coupled to an internal cavity of the liquid cell. The liquidcell may initially have fluid contained in at least a portion of theliquid cell. An excitation voltage is applied to a drive element such asa piezoelectric drive element disposed on the cantilevered probe. Thedrive element may comprise a material such as, for example, zinc oxide,lead zirconate titanate, polyvinylidene fluoride, or a piezoelectricfilm. The drive element may be passivated to electrically isolate thedrive element when a conductive fluid is placed in the internal cavityof the liquid cell. The drive element may be passivated with a materialsuch as silicon nitride, silicon dioxide, parylene, Teflon, aninsulating polymer, or an insulating material. The excitation voltage isapplied to the drive element through one or more conductive feedthroughsthat are connected between the internal cavity and a dry side of theliquid cell. The cantilevered probe may be driven at a frequency at ornear a resonant frequency of the cantilevered probe. The cantileveredprobe may be driven into oscillation at its fundamental resonantfrequency, for example, or at a higher resonant frequency correspondingto a higher order resonant mode. The cantilevered probe may be driven,for example, at or near a resonant frequency or at an off-resonancefrequency. The amplitude of oscillation may be controlled, for example,by controlling the amplitude of the drive signal applied to the driveelement.

In a tapping mode that uses a mechanical stop, the atomic probe tipcoupled to a free end of the cantilevered probe is tapped against areference surface or mechanical stop, as seen at block 520. Themechanical stop is coupled to a base end of the cantilevered probe. Theatomic probe tip is tapped against the mechanical stop in the tappingmode to provide a reference tapping amplitude that shifts, for example,when a target chemical or biological species is applied to a treatedsection of the cantilevered probe. The cantilevered probe or themechanical stop may be positioned such that the free end of thecantilevered probe lightly strikes the mechanical stop, or such that anatomic probe tip attached near the free end of the cantilevered probelightly strikes the mechanical stop. The tapping amplitude can beadjusted, for example, by adjusting the drive voltage to the driveelement, or by positioning the mechanical stop with a positioningelement coupled between the mechanical stop and the liquid cell.

The mechanical stop typically comprises a smooth, relatively hardsurface for the cantilevered probe to tap. For example, a portion of apolished silicon wafer, a smooth glass plate, a mica surface, a smoothalumina surface, or a ground and polished metal plate may form thecontact surface of the mechanical stop. The mechanical stop may includea positioning element coupled between the mechanical stop and the baseend of the cantilevered probe.

The positioning element provides the ability to position the mechanicalstop at a suitable location for striking by the cantilevered probe.Translational movements in one or more directions may be provided withtranslation actuators such as screw drives, linear actuators, orpiezoelectric actuators. The mechanical stop may be coarsely adjusted bythe positioning element to engage the cantilevered probe in a tappingmode. The mechanical stop may be positioned with fine adjustments of thepositioning element, which are used to maintain an oscillation of thecantilevered probe at a nominally constant amplitude. The positioningelement may comprise, for example, a piezotube or other suitablepositioning mechanism for providing fine positioning of the mechanicalstop with respect to the cantilevered probe. Alternatively, thepositioning element may be coupled directly to the base end of thecantilevered probe. The mechanical stop and at least a portion of thepositioning element may be enclosed by or may be in close proximity tothe liquid cell.

The cantilevered probe is driven into oscillation with a drive element,and the oscillating cantilevered probe is tapped against the mechanicalstop or reference surface. Amplitudes of oscillation are measured, andchanges in the oscillation amplitude are measured after exposing thecantilevered probe to a target biological or chemical species. Thetarget species is determined based on oscillation amplitudes of thecantilevered probe that are measured by the sense element when a treatedsection of the cantilevered probe is exposed to the target chemical orbiological species.

A first amplitude or frequency is measured with a sense element that iscoupled to the cantilevered probe, as seen at block 530. The senseelement may comprise, for example, an optical sense element, apiezoelectric sense element, a piezoresistive sense element, acapacitive sense element, or a magnetic sense element. The sense elementmay comprise, for example, a piezoresistive sense element coupled to thecantilevered probe. The deflection amplitude or frequency may bemeasured, for example, by directing a beam of light from a light sourceonto a surface near the free end of the oscillating cantilevered probeand detecting the beam of light when the beam of light is reflected fromat least a portion of the oscillating cantilevered probe, such as with aphotodetector or a photodetector array.

The sense element may comprise, for example, a piezoelectric senseelement disposed on the cantilevered probe. The deflection amplitude orfrequency of the oscillating cantilevered probe is measured by drivingthe cantilevered probe into oscillation with a piezoelectric drive-senseelement mounted on the cantilevered probe, and measuring a signal fromthe piezoelectric drive-sense element when the cantilevered probe isoscillating. In another example, the cantilevered probe may be broughtinto intermittent contact with the reference surface. Theroot-means-square (rms) amplitude of the AC voltage generated by thepiezoelectric sense element is proportional to the oscillation ordeflection amplitude of the cantilever. When the cantilevered probe iscontacting the surface intermittently, the oscillation amplitude can becompared to a set-point amplitude and maintained at a specified value bya feedback loop through the drive circuit. Exposing the cantileveredprobe to a target chemical or biological species causes the cantileveredprobe to bend up and away from the reference surface on which it taps.The feedback loop responds by moving the surface vertically with apiezotube or other positioning element until the specified cantileveredamplitude is restored. The piezotube response may be monitored withdata-acquisition hardware and software.

The cantilevered probe may be straight initially or have some minorcurvature due to processing or due to effects of the treated section ona surface of the cantilevered probe. When the treated section isexposed, for example, to a chemical or biological species that reactswith it, the treated section may expand or contract, causing bending ofcantilevered probe and a change in the neutral position. For example,the treated section located near a base end of the cantilevered probemay enlarge when exposed to a target species, and cause a slightcurvature of the cantilevered probe, which results in a deflection shiftat the free end of the probe. Slowly changing or quasi-staticdisplacements of the cantilevered probe occur when deflections of thecantilevered probe change slowly with exposure to a chemical orbiological species or with slow changes in the concentration of thechemical species in the measurement medium. Changes are considered slowwhen the time frame for the change is longer than the period ofvibration of the oscillating cantilevered probe.

Static displacements may be measured optically or with strain-sensitivepiezoelectric or piezoresistive elements positioned on the cantileveredprobe. Static displacements can be measured electrostatically ormagnetically. Generally, static displacements are difficult to measurewith piezoelectric sense elements because of the gradual decrease orbleed-off of charge generated when the piezoelectric material isstrained. However, dynamic displacements are readily measured withpiezoelectric films, particularly when the frequency of vibration isappreciably faster than the decay rate of generated charge. Anoscillating or vibrating cantilevered probe creates a charge that can bedetected, for example, with a charge amplifier, a transimpedanceamplifier, or an AC bridge circuit that generates an output voltage,which provides a measure of the deflection amplitude. The output voltagecan also be used to measure a frequency of one or more resonant modes ofthe cantilevered probe when the cantilevered probe is oscillated. Staticor quasi-static displacements of the neutral position of thecantilevered probe that are due to chemical or biological exposure,however, are difficult to measure with a piezoelectric sense elementeven with dynamic oscillations unless there is a reference surface.

A reference surface may be provided by a mechanical stop. The mechanicalstop is initially positioned such that the free end of the cantileveredprobe or a probe tip coupled to the free end of the cantilevered probestrikes the stop when the probe is oscillating. The mechanical stop maybe positioned, for example, such that the cantilevered probe lightly orheavily taps the mechanical stop.

A fluid may be injected into the interior cavity of the liquid cellthrough an inlet port connected between the internal cavity and the dryside of the liquid cell, as seen at block 540. The fluid may be added toor replace other fluid in the interior cavity of the liquid cell. Thefluid may include the target species. The fluid may comprise a liquid ora gas, and may be forcibly or diffusively injected into the interiorcavity. An outlet port may be used to vent, evacuate, or recirculate thefluid in the interior cavity.

A treated section of the cantilevered probe is exposed to a targetspecies, as seen at block 550. The cantilevered probe may gain or losemass, for example, when the treated section of the cantilevered probe isexposed to the target species. Alternatively, the treated section whenexposed to the target species may cause an upward or downward bending ofthe cantilevered probe that may be detected by various types ofmeasurements. The measurements include the static or quasi-staticdeflection of the cantilevered probe, the amplitude of a probe tiptapping against a reference surface or mechanical stop, the voltageapplied to a piezotube supporting the cantilevered probe or coupled tothe mechanical stop, the drive voltage required to keep the cantileveredprobe in minimal contact with the mechanical stop, and other suitablemeasurements techniques for determining the quasi-static bending of thecantilevered probe.

The target species may include, for example, mercury, hydrogen, analcohol, water vapor, a chemical element, a chemical compound, anorganic material, an inorganic material, a biological material, a DNAstrand, a bioactive agent, or a toxin. The treated section of thecantilevered probe may comprise, for example, a patterned gold layer, apalladium layer, an alcohol-absorbent polymer, a water-absorbentmaterial, a chemical-sensitive layer, a biosensitive material, or athiol. The treated section of the cantilevered probe may be, forexample, on the topside or underside of the cantilevered probe. When thetreated section of the cantilevered probe is exposed to the chemical orbiological species, the treated section may react or respond byincreasing or lessening the stiffness of the cantilevered probe, whichchanges the resonant frequency of the cantilevered probe. The resonantfrequency can also be changed when the exposure of the treated sectionto a chemical or biological species causes the treated section to addmass to or subtract mass from the cantilevered probe. The treatedsection when exposed to the chemical or biological species could alsocause the cantilevered probe to bend or curve quasi-statically from itsinitial neutral position, or some combination thereof.

A second deflection or frequency of the cantilevered probe is measured,as seen at block 560. The second deflection amplitude or frequency isgenerally determined with the same sense element as used for the firstdeflection or frequency measurement.

The deflection amplitude or frequency of the oscillating cantileveredprobe may be impacted by the interaction between the chemical orbiological species and the treated section of the cantilevered probe.The fundamental resonant frequency of the oscillating cantilevered probemay be measured, or a suitable overtone such as the second or third modemay be measured. Oscillation frequencies may be determined fromamplitude measurements taken as a function of time. Phase informationmay also be extracted from the amplitude information by comparing theamplitude information to reference signals from, for example, the drivesignal.

The target species is determined based on the first deflection amplitudeor frequency measurement and the second deflection amplitude orfrequency measurement, as seen at block 570. The presentation andconcentration of the target species may be determined, for example, bymeasuring the amount of frequency shift of the cantilevered probe. Thepresentation and concentration of the target species may be determined,for example, by determining the magnitude of the static or quasi-staticbending of the cantilevered probe. In one example, the chemical orbiological species may be determined to be absent or present. In anotherexample, the concentration of the species may be determined. In anotherexample, a particular type of chemical or biological species may bedetected and quantified according to the specificity of the treatedsection. The target chemical or biological species may be determined,for example, based on the first deflection amplitude and the seconddeflection amplitude when operating in an open-loop mode. In anotherexample, the chemical or biological species may be determined based onthe position of the mechanical stop when operating in a closed-loopmode. In another example, the chemical or biological species may bedetermined based on the measured frequency of the oscillatingcantilevered probe, such as the fundamental frequency or an overtone.

The chemical or biological species may be determined continuously, forexample, when the treated section responds continuously to thecomposition and the concentration of the exposed target species. Inanother example, the concentration of the target species may bedetermined at its peak value when the treated section of thecantilevered probe does not respond reversibly after the target speciesis applied and then withdrawn. In this case, a new probe may be requiredor the old one may need to be cleaned.

Temperature variations within the enclosure may result in staticbeam-bending of the cantilevered probe, particularly when two dissimilarmaterials with differing thermal expansion coefficients are used to formthe cantilevered probe. Compensation of beam-bending due to temperaturemay be accomplished by combining, for example, measurements from aseparate, non-exposed cantilevered probe, an untreated cantileveredprobe, or a temperature measuring device such as a resistive temperaturedevice (RTD), thermocouple, or diode-based temperature sensor mounted onor in the liquid cell.

The cantilevered probe may be heated to initialize or re-initialize thetreated section of the cantilevered probe, so that a new reading or anaccurate first reading may be made. The cantilevered probe may bere-initialized, for example, by reversing any chemical reactions thathave occurred at the treated section. The cantilevered probe may becoupled to and heated by a resistive heater coupled to the cantileveredprobe. Alternatively, an external heater such as a heat lamp or a hotgas system may be used to heat and re-initialize the cantilevered probe.Chemical re-initialization may be accomplished, for example, by usingcleaning processes such as wiping or solvent exposure, or by reversingany chemical reactions that occurred to the treated section. Thecantilevered probe may be cleaned, for example, with successive dips inacetone, ethanol, or with an oxygen plasma.

While the embodiments of the invention disclosed herein are presentlyconsidered to be preferred, various changes and modifications can bemade without departing from the spirit and scope of the invention. Thescope of the invention is indicated in the appended claims, and allchanges that come within the meaning and range of equivalents areembraced herein.

1. A method of sensing a target species with a cell, comprising:providing a cell comprising a cantilevered probe coupled to an internalcavity of the cell; driving the cantilevered probe with an excitationvoltage, wherein the excitation voltage is applied to a piezoelectricelement on the cantilevered probe through at least one conductivefeedthrough connected between the internal cavity and an outer surfaceof the cell; measuring a first deflection amplitude of the cantileveredprobe with the piezoelectric element; exposing a treated section of thecantilevered probe to a target species; measuring a second deflectionamplitude of the cantilevered probe; and determining the target speciesbased on the first deflection amplitude and the second deflectionamplitude.
 2. The method of claim 1 wherein the piezoelectric element isat least partially made from one or more materials selected from thegroup consisting of zinc oxide, lead zirconate titanate, polyvinylidenefluoride, and a piezoelectric film.
 3. The method of claim 1 wherein thepiezoelectric element is passivated to electrically isolate thepiezoelectric element when a conductive fluid is placed in the internalcavity of the cell.
 4. The method of claim 1 wherein the piezoelectricelement is passivated with a material selected from the group consistingof silicon nitride, silicon dioxide, parylene, Teflon, an insulatingpolymer, and an insulating material.
 5. The method of claim 1 whereinthe cantilevered probe is driven at a frequency near a resonantfrequency of the cantilevered probe.
 6. The method of claim 1 furthercomprising: measuring a first frequency of the cantilevered probe withthe piezoelectric element coupled to the cantilevered probe; exposingthe treated section of the cantilevered probe to the target species;measuring a second frequency of the cantilevered probe; and determiningthe target species based on the first frequency measurement and thesecond frequency measurement.
 7. The method of claim 1 furthercomprising injecting a fluid into the interior cavity of the cellthrough an inlet port connected between the internal cavity and an outersurface of the cell, the fluid comprising the target species.
 8. Themethod of claim 1 further comprising tapping an atomic probe tip coupledto a free end of the cantilevered probe against a mechanical stop, themechanical stop coupled to a base end of the cantilevered probe.
 9. Themethod of claim 1 wherein the cantilevered probe is driven at a higherorder resonant mode.
 10. The method of claim 1 further comprisingsensing cantilever bending based on measuring a second deflectionamplitude of the cantilevered probe.
 11. The method of claim 1 furthercomprising compensating for temperature variation induced cantileverdeflection.
 12. The method of claim 11, wherein the cantilevered probecomprises a resistive heater, further comprising heating the resistiveheater to compensate for temperature variation induced cantileverdeflection.
 13. The method of claim 1, wherein the cantilevered probecomprises a resistive heater, further comprising heating the resistiveheater to initialize or clean the cantilevered probe.
 14. The method ofclaim 1, wherein the cantilevered probe comprises a resistive heater,further comprising heating the resistive heater to react a targetchemical species.
 15. The method of claim 1, wherein the cantileveredprobe is one of a plurality of cantilevered probes in a cantileveredprobe array.
 16. The method of claim 15, wherein each of the pluralityof cantilevered probes comprises a resistive heater.
 17. The method ofclaim 16, further comprising independently heating at least a portion ofthe resistive heaters of the plurality of cantilevered probes.